HIV protease inhibitors are important pharmacological agents used in the treatment of HIV-infected patients. One of the major disadvantages of HIV protease inhibitors is that they increase several cardiovascular risk factors, including the expression of CD36 in macrophages. The expression of CD36 in macrophages promotes the accumulation of cholesterol, the development of foam cells, and ultimately atherosclerosis. Recent studies have suggested that α-tocopherol can prevent HIV protease inhibitor-induced increases in macrophage CD36 levels. Because of the potential clinical utility of using α-tocopherol to limit some of the side effects of HIV protease inhibitors, we tested the ability of α-tocopherol to prevent ritonavir, a common HIV protease inhibitor, from inducing atherosclerosis in the LDL receptor (LDLR) null mouse model. Surprisingly, α-tocopherol did not prevent ritonavir-induced atherosclerosis. However, cotreatment with the nucleoside reverse transcriptase inhibitors (NRTIs), didanosine or D4T, did prevent ritonavir-induced atherosclerosis. Using macrophages isolated from LDLR null mice, we demonstrated that the NRTIs prevented the upregulation of CD36 and cholesterol accumulation in macrophages. Treatment of LDLR null mice with NRTIs promoted the ubiquitination and downregulation of protein kinase Cα (PKC). Previous studies demonstrated that HIV protease inhibitor activation of PKC was necessary for the upregulation of CD36. Importantly, the in vivo inhibition of PKC with chelerythrine prevented ritonavir-induced upregulation of CD36, accumulation of cholesterol, and the formation of atherosclerotic lesions. These novel mechanistic studies suggest that NRTIs may provide protection from one of the negative side effects associated with HIV protease inhibitors, namely the increase in CD36 levels and subsequent cholesterol accumulation and atherogenesis.
- low-density lipoprotein receptor
highly active antiretroviral therapy (HAART) is essential to decreasing the morbidity and mortality of HIV-infected individuals (10, 22, 27). However, prolonged use of HAART is associated with hyperlipidemia, lipodystrophy, insulin resistance, and accelerated atherosclerosis (7, 10, 11, 17, 26). Of the various classes of compounds used in HAART, the HIV protease inhibitors appear to be associated with negative cardiovascular risk factors (7, 10, 11, 17, 26). Because of the importance of HIV protease inhibitors in HAART, it is crucial to understand the mechanisms whereby HIV protease inhibitors induce cardiovascular side effects.
HIV protease inhibitors affect multiple cellular mechanisms, including inhibiting the degradation of the nuclear form of sterol regulatory element binding proteins (nSREBP) in the liver and adipose tissue (2, 15, 24). Accumulation of nSREBP in the liver increases fatty acid and cholesterol biosynthesis whereas in adipose tissue accumulation of nSREBP induces lipodystrophy, insulin resistance, and decreases leptin levels (2, 15, 24). HIV protease inhibitors also decrease the degradation of nascent apolipoprotein B by suppressing proteasome activity (16). Insulin resistance and diabetes are promoted by the ability of HIV protease inhibitors to attenuate glucose transporter GLUT4 activity in muscle and adipose tissues (12).
In addition to the above effects, Dressman et al. (5) demonstrated that HIV protease inhibitors contribute to the formation of atherosclerosis by promoting the upregulation of CD36 and the subsequent accumulation of sterol in macrophages. CD36 is a class B scavenger receptor that facilitates the uptake of modified lipoproteins into macrophages, which promotes foam cell formation and the development of atherosclerotic lesions. The use of CD36 blocking antibodies, CD36 antisense morpholino oligonucleotides, and CD36 null mice, clearly demonstrated that CD36 upregulation was necessary for HIV protease inhibitor-induced sterol accumulation in macrophages and atherosclerotic lesion formation (5). The fundamental finding that HIV protease inhibitors increase the amount of CD36 in macrophages has been confirmed by Munteanu et al. (19).
The reported mechanism for HIV protease inhibitor upregulation of CD36 involved the upregulation of peroxisome proliferator-activated receptor-γ (PPARγ) (5). PPARγ has been previously reported to be upregulated by molecules found in modified lipoproteins such as oxidized low-density lipoprotein (6, 20). Importantly, the upregulation of PPARγ by HIV protease inhibitors and modified lipoproteins leads to increased CD36 expression, which then promotes further uptake of modified lipoproteins, foam cell formation, and subsequently atherosclerotic lesions (6, 20). HIV protease inhibitors stimulated the expression of PPARγ via protein kinase C, but the entire molecular pathway has not been elucidated (5, 6). In contrast, Munteanu et al. (19), using THP-1 monocytes, suggested that the primary mechanism for increasing the amount of CD36 in macrophages is due to the inhibition of the proteasome. This conclusion was based on the use of the proteasome inhibitor N-acetyl-Leu-Leu-norleucinal (ALLN), which significantly increased CD36, whereas only a modest increase in CD36 mRNA was observed in the presence of the HIV protease inhibitor ritonavir (19). Presently it is unclear how many mechanisms contribute to HIV protease inhibitor-induced increases in the level of CD36 and the relative importance of each mechanism.
Importantly, the study by Munteanu et al. (19) detailed a finding of potentially great clinical significance, namely that α-tocopherol, in THP-1 monocytes, prevented ritonavir-induced increases in CD36 levels by preventing the inhibition of proteasome activity. If α-tocopherol prevents HIV protease inhibitor-induced increases in CD36 levels in humans, a simple and safe treatment would be available to counteract at least one of the negative side effects of HIV protease inhibitors. While the cell culture studies done by Munteanu et al. (19) are intriguing, the in vivo effects of α-tocopherol have not been determined. In the present study we tested the ability of α-tocopherol to prevent HIV protease inhibitors from increasing CD36 levels in an animal model. As a control, we used nucleoside analog reverse transcriptase inhibitors (NRTIs), which are commonly used in combination therapy with HIV protease inhibitors. To our surprise, α-tocopherol did not prevent HIV protease inhibitor-induced increases in CD36 or the development of atherosclerotic lesions. However, NRTIs completely prevented the upregulation of CD36 and the development of atherosclerosis.
MATERIALS AND METHODS
RPMI medium 1640, DMEM high-glucose medium, fetal calf serum, l-glutamine, trypsin-EDTA, and penicillin-streptomycin were purchased from Life Technologies (Grand Island, NY). Percoll, polyvinylidene difluoride (PVDF) membranes, Tween 20, α-tocopherol, and chelerythrine chloride were purchased from Sigma (St. Louis, MO). Bradford reagent was purchased from Bio-Rad (Hercules, CA). The anti-PKCα IgG was from BD Biosciences (San Diego, CA), the anti-actin IgG was from Sigma, the anti-mouse CD36 (IgM) was from BioDesign International (Kennebunk, ME), the anti-SRA (scavenger receptor, type A) was from Serotec, anti-ubiquitin was from Zymed Laboratories (San Francisco, CA), and Quality Control Biochemicals (St. Louis, MO) generated the SR-BI (scavenger receptor, type B, class I) antibody as a fee for service. Horseradish peroxidase-conjugated IgGs were supplied by Cappel (West Chester, PA). Super Signal chemiluminescent substrate was purchased from Pierce (Rockford, IL). Bristol-Myers Squibb provided the didanosine and Abbott Laboratories provided the ritonavir. The mouse feed was obtained from Harlan Tekland (Madison, WI). The protein kinase C assay kit was from Calbiochem (San Diego, CA). [γ-32P]adenosine 5′-triphosphate (109 TBq/mmol) was from Perkin Elmer (Boston, MA). The ubiquitin enrichment kit was from Pierce.
Sample buffer (5×) consisted of 0.31 M Tris·HCl, pH 6.8, 2.5% (wt/vol) SDS, 50% (vol/vol) glycerol, and 0.125% (wt/vol) bromophenol blue. Tris-buffered saline (TBS) consisted of 20 mM Tris·HCl, pH 7.6, and 137 mM NaCl.
All animals were housed in the University of Kentucky animal facilities. Animals were maintained in constant temperature conditions on a 14:10-h light/dark cycle (lights on at 0400 h), and they were provided food and water ad libitum. The LDLR null mice were obtained from The Jackson Laboratory (Bar Harbor, ME). At 6 wk of age mice were placed on a chow diet containing 0, 400, or 800 mg/kg of α-tocopherol and given either vehicle control (0.01% ethanol), ritonavir (50 μg/day), didanosine (75 μg/day), or D4T (75 μg/day) in the drinking water. Where indicated, chelerythrine chloride (5 mg/kg) was injected intraperitoneally every 48 h for the duration of the study (3). This regimen of ritonavir has previously been described as producing atherosclerotic lesions in male LDLR null mice without altering plasma cholesterol levels (5). Mouse peritoneal macrophages were obtained by lavage (29).
Cholesterol and cholesteryl ester mass quantification.
Cholesterol and cholesteryl ester mass was quantified using a commercially available kit from Wako Chemicals (Richmond, VA) per manufacturer's instructions.
SDS-PAGE and immunoblotting.
Samples were concentrated by trichloroacetic acid precipitation and washed in acetone. Pellets were suspended in sample buffer that contained 1.2% (vol/vol) β-mercaptoethanol (Laemmli sample buffer) and heated at 95°C for 3 min before being loaded into gels. Proteins were separated in 12.5% SDS-polyacrylamide gels by using the method of Laemmli (13). The separated proteins were then transferred to PVDF membranes. Membranes were blocked in TBS + 5% dry milk for 1 h at room temperature. Primary antibodies were diluted in TBS + 1% dry milk and incubated with membranes for 1 h at room temperature. Membranes were then washed four times, 10 min per wash, in TBS + 1% dry milk. The secondary antibodies (all conjugated to horseradish peroxidase) were diluted 1/20,000 in TBS + 1% dry milk and incubated with membranes for 1 h at room temperature. The membranes were then washed in TBS, and the bands were visualized by chemiluminescence.
Northern blot hybridization.
Total RNA was extracted from cells using TRIzol (Invitrogen, Carlsbad, CA). RNA was quantified by spectrometry, and 20 μg of total RNA was loaded into each lane of a 5% denaturing agarose gel. Following electrophoretic separation, RNA was transferred to nylon membranes and probed for PPARγ, PKCα, and GAPDH mRNAs, as previously described (5).
Protein A-Sepharose beads were first blocked by incubation for 4 h at 4°C with a peritoneal macrophage cell lysate (200 μg/ml), plus 30 mg/ml of BSA in immunoprecipitation buffer (150 mM NaCl, 0.5% Triton X-100, 50 mM Tris·HCl, pH 8.0). Blocked beads were then used to preclear the experimental fractions that had been adjusted to 0.5% (vol/vol) Triton X-100. Precleared fractions were incubated for 2 h at 4°C with the appropriate antibody before adding blocked Protein A-Sepharose beads and incubating an additional 1 h at 4°C. The beads were collected by centrifugation, washed four times in high-salt (500 mM NaCl) immunoprecipitation buffer, and finally treated with Laemmli sample buffer. Immunoprecipitated proteins were detected by Western blotting.
Quantification of atherosclerotic lesions.
After 8 wk of treatment, plasma was collected for cholesterol, cholesteryl ester, and triglyceride determinations. The mice were then processed to quantify the surface area of atherosclerotic lesions. Atherosclerotic lesions were quantified as we have done previously (4, 5). Briefly, the aorta from the arch to the ileal bifurcation was collected, the extraneous tissue was dissected away, and the intimal surfaces were exposed by a longitudinal cut. The aortas were placed under a dissecting microscope equipped with a charge-coupled device camera attachment that captures the image directly to a computer file. Atherosclerotic lesions on the intimal aortic surface appear as bright white areas, compared with the thin and translucent aorta. Areas of intima covered by atherosclerosis were quantified with ImagePro software 6.0 (Media Cybernetics, Silver Spring, MD).
Least squares analysis of variance was used to evaluate the data with respect to sample, treatment, time, and their interactions, using the ANOVA procedure of Statistica (StatSoft, Tulsa, OK). When appropriate, samples were compared using the Tukey HSD test. Means were considered different at P < 0.01.
Effect of α-tocopherol on ritonavir-induced atherosclerosis.
Munteanu et al. (19) have reported that α-tocopherol can prevent HIV protease inhibitor-induced accumulation of cholesterol in THP-1 macrophages. These in vitro data suggested that α-tocopherol may be effective at preventing or reducing HIV protease inhibitor-induced atherosclerosis. To test whether α-tocopherol can prevent the atherogenic effect of ritonavir, 6-wk-old male LDLR null mice fed a standard chow diet were treated with ritonavir (50 μg/day), vehicle control (0.01% ethanol), α-tocopherol (400 mg/kg), or ritonavir (50 μg/day) and α-tocopherol (400 mg/kg or 800 mg/kg) in their drinking water for 8 wk, as we described previously (5). LDLR null mice were used for these studies because this mouse model develops few atherosclerotic lesions in the absence of a high-fat diet or HIV protease inhibitors, as we previously demonstrated (5). Thus the effects of ritonavir and α-tocopherol on atherosclerotic lesions can be studied independently of other factors that affect lesion formation (i.e., serum cholesterol). At the conclusion of the study, the ascending and descending aortas were removed and opened, and the areas covered by atherosclerotic lesions were quantified by image analysis (4, 5) (Fig. 1). Animals treated with vehicle or α-tocopherol did not have significant lesions, whereas animals treated with ritonavir developed significant lesions, as previously demonstrated by Dressman et al. (5). Animals treated with ritonavir and α-tocopherol developed atherosclerotic lesions to the same extent as animals treated with ritonavir only. Importantly, two doses of α-tocopherol (400 and 800 mg/kg) did not protect animals from ritonavir-induced atherosclerotic lesion formation.
Effect of NRTIs on ritonavir-induced atherosclerosis.
In HAART, patients are often treated with protease inhibitors, in combination with other anti-retroviral drugs, such as nucleoside reverse transcriptase inhibitors, to increase the efficacy of anti-retroviral activity. Since we predicted that α-tocopherol would decrease protease inhibitor-induced atherosclerotic lesion size, we also used two different NRTIs, didanosine and D4T, presumably used as negative controls that would not affect ritonavir-induced atherosclerosis. Six-week-old male LDLR null mice fed a standard chow diet were treated with didanosine (75 μg/day), D4T (75 μg/day), ritonavir (50 μg/day) and didanosine (75 μg/day), or ritonavir (50 μg/day) and D4T (75 μg/day) in the drinking water for 8 wk. Animals treated with didanosine or D4T alone did not develop significant atherosclerotic lesions (Fig. 1). Surprisingly, animals treated with ritonavir and didanosine, or ritonavir and D4T did not develop atherosclerotic lesions. To our knowledge these are the first data that indicate that NRTIs can limit the atherogenic effects of ritonavir.
NRTIs prevent an increase in macrophage cholesterol and CD36 levels.
Figure 1 clearly demonstrates that didanosine and D4T can protect mice from ritonavir-induced atherosclerosis. Thus we next investigated the mechanism whereby protection is afforded by NRTIs. We previously demonstrated that ritonavir induces the accumulation of cholesterol in macrophages and that cholesterol accumulation and atherosclerotic lesion formation were dependent on the upregulation of CD36 (5). To determine whether didanosine and D4T affected macrophage cholesterol accumulation and CD36 protein levels, peritoneal macrophages were isolated from mice treated as described in Fig. 1. Consistent with previous studies (5), ritonavir increased the cholesterol associated with macrophages (Fig. 2A) by two- to threefold. Didanosine and D4T alone did not alter cholesterol levels compared with the vehicle control. However, didanosine and D4T both inhibited ritonavir-induced increases in macrophage cholesterol. Consistent with Fig. 1, α-tocopherol did not affect macrophage cholesterol levels or prevent ritonavir-induced increases in macrophage cholesterol. Figure 2B illustrates that didanosine and D4T prevented ritonavir upregulation of CD36 in macrophages. Again, α-tocopherol was ineffective at preventing ritonavir-induced upregulation of CD36. Although it has been established that ritonavir induces the generation of atherosclerotic lesions in LDLR null mice by the upregulation of CD36 (5), other proteins may affect lesion development. To determine whether didanosine or D4T was altering other atherogenic proteins, we examined the levels of SR-BI and SRA (Fig. 2B). Importantly, none of the treatments altered the levels of SR-BI and SRA, suggesting that these proteins were not involved in the protective effects of didanosine and D4T. Figure 2B shows the actin loads from the described samples, indicating that equal protein was loaded into each well. These data suggest that the mechanism whereby didanosine and D4T prevented HIV protease inhibitor-induced atherosclerosis was by preventing the upregulation of CD36 in macrophages.
NRTIs promoted the loss of PKCα protein in macrophages.
We previously used molecular and genetic models to demonstrate that ritonavir-induced CD36 upregulation was mediated by a PKC-dependent increase in PPARγ, which subsequently increased the expression of CD36 (5). To determine whether didanosine and D4T prevented ritonavir-induced CD36 upregulation, and atherosclerotic lesion formation by a PPARγ mechanism, we isolated RNA from peritoneal macrophages obtained from LDLR null mice, treated as described for Fig. 1. Ritonavir increased PPARγ mRNA levels without altering PKCα mRNA levels (Fig. 3A). Treatment with α-tocopherol did not inhibit ritonavir-induced upregulation of PPARγ, in agreement with Figs. 1 and 2. Importantly, didanosine and D4T prevented ritonavir-induced upregulation of PPARγ mRNA. Since PKCα mRNA levels were not changed, we next examined the levels of PKCα protein. Figure 3B demonstrates that didanosine and D4T in the absence or presence of ritonavir promoted the loss of PKCα protein, whereas animals treated with α-tocopherol had normal levels of PKCα. These data demonstrated that didanosine and D4T caused the loss of PKCα, which may be responsible for the inhibition of PPARγ upregulation.
NRTIs promoted the ubiquitination of PKCα.
The data shown in Fig. 3 demonstrated that didanosine and D4T promoted the loss of PKCα protein, without affecting the level of PKCα mRNA, which suggested that a post-translational mechanism is responsible for the loss of PKCα protein. A common mechanism for promoting cytosolic protein degradation is the ubiquitination of proteins, so that they are targeted to the proteasome. To determine whether ubiquitination was involved in the loss of PKCα, we immunoprecipitated PKCα from macrophages isolated from animals treated as described in Fig. 1. Each immunoprecipitation was done with 500 μg of cell protein, whereas only 20 μg of protein was analyzed in Fig. 3, which likely accounts for the apparent lack of PKCα in Fig. 3, and the presence of a small amount of PKCα in Fig. 4. The precipitated material was then resolved by SDS-PAGE and immunoblotted by using an anti-ubiquitin antibody. The amount of cross-reactive material was quantified by image analysis, normalized to the amount of actin in the original immunoprecipitation sample and plotted in Fig. 4A. The data demonstrate that didanosine and D4T dramatically increased the amount of PKCα that has undergone ubiquitination. Importantly, the vehicle, ritonavir, and α-tocopherol samples did not affect PKCα ubiquitination. The non-NRTI-treated samples contained ∼90-fold more PKCα than the NRTI-treated samples but had 5–8-fold less ubiquitinated PKCα (Fig. 4B). These data suggest that didanosine and D4T prevented CD36 upregulation by promoting the degradation of PKCα.
In vivo inhibition of PKC prevents HIV protease inhibitor-induced increases in macrophage CD36.
Collectively, the data presented thus far suggest that didanosine and D4T protected against ritonavir-induced atherosclerosis by downregulating PKCα and consequently preventing the upregulation of CD36. To determine whether the lack of functional PKCα can prevent ritonavir-induced atherosclerosis, we treated 6-wk-old LDLR null mice with 2.5 mg/kg chelerythrine, a PKC inhibitor, along with ritonavir (50 μg/day) for 8 wk. Peritoneal macrophages were isolated from the mice at the conclusion of the study, and the extent of PKC activity was determined with a commercially available enzymatic assay system. Figure 5A demonstrates that ritonavir increased PKC activity sixfold above the vehicle control. Importantly, chelerythrine decreased basal PKC activity and ritonavir-induced PKC activity, thereby demonstrating the effectiveness of chelerythrine at inhibiting macrophage PKC. Consistent with a decrease in PKC activity, chelerythrine prevented ritonavir-induced upregulation of PPARγ (Fig. 5B) and an increase in the amount of CD36 protein (Fig. 5C). These data support the concept that PKC is necessary for ritonavir-induced increases in CD36 protein levels.
In vivo inhibition of PKCα prevents HIV protease inhibitor-induced atherosclerotic lesions.
The data suggested that didanosine and D4T inhibit ritonavir-induced atherosclerosis by causing the ubiquitination and downregulation of PKCα. The PKC inhibitor chelerythrine prevented the upregulation of PPARγ and CD36 in macrophages. To determine whether the inhibition of PKC activity protected against ritonavir-induced atherosclerosis, LDLR null mice were treated with chelerythrine as described in Fig. 5. Figure 6A demonstrates that chelerythrine prevented the accumulation of cholesterol in macrophages from mice treated with both ritonavir and chelerythrine. Dramatically, chelerythrine, like didanosine and D4T, completely inhibited ritonavir-induced atherosclerosis (Fig. 6B). These data demonstrate that NRTI-induced depletion of PKC can account for the ability of NRTIs to prevent HIV protease inhibitor-induced atherosclerosis.
The development of aspartyl endopeptidase protease inhibitors, which catalyze the cleavage of the HIV gag and gag-pol polyproteins, has been one of the most significant advances in HIV therapy (10, 22, 27). The usefulness of HIV protease inhibitors is tempered by significant side effects, including lipodystrophy, hyperlipidemia, visceral adiposity, and insulin resistance (7, 10, 11, 17, 26). The ability of HIV protease inhibitors to interfere with the function of the proteasome results in an increase in sterol regulatory element binding proteins (SREBPs) and apoB, both of which may contribute to lipodystrophy, hyperlipidemia, and cardiovascular diseases, such as atherosclerosis (7, 10, 11, 17, 26). However, a study by Dressman et al. (5) demonstrated that in mouse models, HIV protease inhibitors can promote atherosclerosis independent of changes in plasma lipid levels. The increase in atherosclerosis was shown to be dependent on the increase in macrophage CD36 protein levels (5). Studies by Munteanu et al. (18, 19) confirmed that HIV protease inhibitors increase macrophage CD36 protein levels. This observation is potentially of clinical significance because reducing the dose of protease inhibitor to lessen the increase in plasma lipids may not correspond to a decrease in risk for cardiovascular disease.
The mechanism(s) whereby HIV protease inhibitors increase CD36 is not completely clear. Dressman et al. (5) reported that HIV protease inhibitors increased PPARγ via a PKC intermediate signaling step. Treatment of macrophages with oxidized LDL has been shown to result in an increase in PPARγ activity and subsequently the expression of CD36; thus the involvement of PPARγ is a plausible mechanism (5, 6). In vitro studies with human peripheral blood monocytes demonstrated that PKC inhibitors prevented ritonavir-mediated upregulation of CD36 (5). In addition, the addition of a PPARγ agonist could overcome the PKC block, suggesting that PKC is upstream of PPARγ (5). In contrast, Munteanu et al. (18, 19) reported that the level of PPARγ mRNA in the monocyte cell line THP-1 was not affected by therapeutic doses of ritonavir. Furthermore, they provided data (18, 19) that inhibition of the proteasome with ALLN increased CD36 levels, and they suggested that ritonavir-induced increases in CD36 are caused by decreased proteasome degradation of CD36. Since different cells and different concentrations of ritonavir were used in the two studies, it is difficult to directly compare the data. However, at the present time both mechanisms are possible, and in fact it is conceivable that both mechanisms may be in part responsible for the increase in macrophage CD36.
Studies by Munteanu et al. (19) also indicated that relatively high levels of α-tocopherol (50 μM) could prevent ritonavir-induced increases in CD36 in THP-1 cells. The authors provided data demonstrating that α-tocopherol could partially relieve the ritonavir-mediated inhibition of the proteasome. Although α-tocopherol has been shown to be ineffective in preventing cardiovascular disease (28), it is entirely possible that α-tocopherol could prevent HIV protease inhibitor-induced atherosclerosis. Consequently, we tested this exciting possibility in our mouse model. To our surprise and disappointment, α-tocopherol, even at a very high concentration, did not prevent ritonavir-mediated atherosclerosis or increases in macrophage CD36 levels. Despite convincing data with THP-1 cells (19), in vivo studies with a mouse model could not reproduce the effect of α-tocopherol on macrophage CD36 protein levels. However, we made the unexpected observation that the NRTIs didanosine and D4T did prevent ritonavir-induced atherosclerosis.
NRTIs are incorporated into the viral genome during replication but are not efficiently removed because the viral reverse transcriptase does not have a proofreading exonuclease activity. NRTIs are a critical component of HAART; however, toxicity has been observed, related to inhibition of mitochondrial DNA replication (14) and fat redistribution syndrome (21). In the current study, treating cells with ritonavir and didanosine, or ritonavir and D4T, presumably was to serve as a negative control for the putative effects of α-tocopherol. Stunningly, the NRTIs prevented ritonavir-induced increases in macrophage CD36 levels and ritonavir-induced atherosclerosis formation. Consistent with Dressman et al. (5), ritonavir induced an increase in PPARγ expression and the NRTIs prevented this ritonavir-induced increase. The NRTIs did not affect the level of PKCα mRNA; however, the NRTIs promoted the loss of PKCα protein in the presence or absence of ritonavir. By using anti-ubiquitin antibodies and immunoprecipitation, we demonstrated that the NRTIs promoted the ubiquitination of PKCα, which led to degradation of the protein by the proteasome. The mechanism by which NRTIs play a role in the ubiquitination of PKCα is not known.
Previous studies by Gogu et al. (8, 9) demonstrated that zidovudine, the first drug in the 2′,3′-dideoxynucleoside class approved by the FDA for treatment of AIDS, prevented erythroid progenitor cell differentiation by inhibiting PKC. Although these investigators did not demonstrate the mechanism of PKC inhibition, their findings are consistent with our didanosine data demonstrating the degradation of PKC. Interestingly, Gogu et al. (9) also demonstrated that α-tocopherol could overcome the inhibitory effect of zidovudine on erythroid progenitor cell differentiation. Although it was not demonstrated, it is possible that α-tocopherol prevented the degradation of PKC. If α-tocopherol prevented the degradation of PKC, based upon our current data, one would predict that animals receiving ritonavir, didanosine, and α-tocopherol would develop atherosclerosis, because didanosine no longer caused the degradation of PKC. The interactions between HIV protease inhibitors, NRTIs, and α-tocopherol are complex and still incompletely understood.
The current study along with the previous study by Dressman et al. (5) strongly points to PKC as being a major regulatory point in mediating the effects of protease inhibitors and NRTIs on the expression of macrophage CD36. To further explore the role of PKC we treated mice with chelerythrine, a PKC inhibitor, in the presence or absence of ritonavir. Since chelerythrine was only given to intact mice, and the peritoneal macrophages were isolated, and were immediately assayed for PKC activity, this indicates that chelerythrine was effective at inhibiting PKC activity in vivo. Chelerythrine was effective at inhibiting PKC phosphorylation activity, inhibiting PPARγ upregulation, and inhibiting increases in CD36 protein levels. Importantly, chelerythrine prevented ritonavir-induced macrophage cholesterol accumulation and ritonavir-induced atherosclerotic lesion formation. It is important to point out that the ability of chelerythrine to prevent atherosclerosis is most likely limited to ritonavir-induced atherosclerosis and not atherosclerosis caused by the other multitude of risk factors.
Although we were unable to confirm the protective effect of α-tocopherol, with regards to ritonavir-induced increases in macrophage CD36, with our in vivo studies, we did discover that NRTIs can prevent an increase in macrophage CD36 levels. Interestingly, NRTIs increased the ubiquitination of PKC, which resulted in the degradation of PKC, and consequently prevented the upregulation of PPARγ, and the increase in macrophage CD36. This mechanism was surprising because it has been suggested that HIV protease inhibitor-related increases in CD36 are due to inhibition of the proteasome (18, 19). However, HIV protease inhibitor effects on the proteasome are complicated. For instance, Schmidtke et al. (25) has demonstrated that ritonavir inhibited the chymotrypsin-like activity of the proteasome but increased the tryptic activity of the proteasome. This finding offers one explanation as to why NRTIs could induce the degradation of PKC, in the presence of HIV protease inhibitors. Alternatively, Piccinini et al. (23) demonstrated that the use of a single protease inhibitor (as we did in the current studies) only had a minimal impact on proteasome activity. In fact, Piccinini et al. (23) showed that the use of three HIV protease inhibitors in combination only caused ∼43% inhibition of the proteasome. Therefore, another possible explanation for our results is that the concentration of ritonavir used in our studies did not sufficiently inhibit the proteasome to affect the degradation of PKC. Finally, Andre et al. (1) have demonstrated that concentrations of ritonavir that do not affect proteasome function can inhibit the presentation of antigen to cytotoxic T lymphocytes; thus it is possible that HIV protease inhibitors and NRTIs impact PKC independent of the proteasome.
The mechanism whereby NRTIs promote the ubiquitination of PKC is unknown. Our current working hypothesis is that HIV protease inhibitors and NRTIs both impact on PKC, and since PKC is upstream of PPARγ, the ability of PPARγ to cause an increase in macrophage CD36 protein levels is increased by HIV protease inhibitors and decreased by NRTIs. This hypothesis does not exclude the contribution of other possible mechanisms nor should it be extended to other non-HIV drug mechanisms for affecting macrophage CD36 protein levels and atherosclerosis.
This work was supported, in part, by National Institutes of Health Grants HL-68509 (to E. J. Smart), DK-63025 (to E. J. Smart), HL-073693 (to M. E. Wilson), and RR-O15592.
We thank the Kentucky Pediatric Research Institute work group for providing invaluable advice and assistance.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2006 the American Physiological Society